The non-radioactive detection of biological analytes utilizing fluorescent labels is an important technology in modern analytical biotechnology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact of reagent disposal is greatly reduced. Examples of methods utilizing such fluorescent detection methods include DNA sequencing, oligonucleotide probe methods, polymerase-chain-reaction product detection, immunoassays, and the like.
One example of analytical equipment employing fluorescent dyes is flow cytometry, a method for detecting, classifying, and characterizing various analytes including biological cells and other particles such as viruses or molecules that exhibit specific characteristics. In a flow cytometer, a flowing liquid stream containing analytes in a liquid medium is directed to an irradiation region. A radiation source, usually a laser, irradiates the analytes, and optical and electronic detection equipment and processors are used to measure spectral properties of the sample, including light absorption, scattering, fluorescence, or phosphorescence to detect the presence of the analytes.
The use of simple fluorescent markers typically entails a purification step to separate bound and unbound fluorescent markers, in order to obtain precise measurements without background interference from the (unbound or bound) group not being measured. Such purification steps add undesired time and expense to what should be a rapid automated process.
In many important applications the independent detection of multiple spatially overlapping analytes in a mixture is required, e.g., single-tube multiplex DNA probe assays, immuno assays, multicolor DNA sequencing methods, and the like. In the case of multi-loci DNA probe assays, by employing spectrally distinguishable fluorescent labels, the number of reaction tubes can be reduced thereby simplifying experimental protocols and facilitating the manufacturing of application-specific kits. In the case of automated DNA sequencing, multicolor fluorescent labeling allows for the analysis of multiple bases in a single lane thereby increasing throughput over single-color methods and reducing uncertainties associated with inter-lane electrophoretic mobility variations.
Multi-color fluorescent detection imposes five severe constraints on the selection of dye labels, particularly for applications requiring a single excitation light source, an electrophoretic separation, and/or treatment with enzymes, e.g., automated fluorescence-based DNA sequencing. First, it is difficult to find a set of dyes whose emission spectra are spectrally resolved, since the typical emission band half-width for organic fluorescent dyes is about 40–80 nanometers (nm) and the width of available spectrum is limited by the excitation light source. Second, even if dyes with non-overlapping emission spectra are found, the set may still not be suitable if the respective fluorescent efficiencies are too low. Third, when several fluorescent dyes are used concurrently, simultaneous excitation becomes difficult because the absorption bands of the dyes are widely separated. Fourth, the charge, molecular size, and conformation of the dyes must not adversely affect the electrophoretic mobilities of the analyte. Fifth, the fluorescent dyes must be compatible with the chemistry used to create or manipulate the analyte, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like. (See, e.g., U.S. Pat. No. 6,008,379.)
One of the limitations of such presently available technologies arises from the available pool of restriction enzymes. These produce a correspondingly limited pool of restriction sites and, therefore, fragments to be detected. Another limitation pertains to the nature of information that is discoverable, i.e., whether the identified target is present, and its relative concentration. It is not, however, readily possible to determine the relative proximity of target sites to each other under fixed or changing environmental conditions.
Fluorescence energy transfer (“FET”) and fluorescence resonance energy transfer (“FRET”) are distance-dependent excited state interactions in which emission of one fluorophore is coupled to the excitation of another. These processes employ two fluorescent dye molecules, one, a donor (D1), having relatively shorter wavelength excitation and emission spectra, and the other dye, an acceptor/reporter (D2), having longer wavelength excitation and emission spectra. Excitation of D1 by a light source of appropriate wavelength ultimately results in emission by D2 at its characteristic wavelength. D1 and D2 must be sufficiently proximate (within about 100 Å) to facilitate energy transfer; this can be accomplished in a number of manners (e.g., via linking to solid supports) or, in the special case of direct fluorescence energy transfer, D1 and D2 can be physically linked together (e.g., through a chain of covalently linked atoms). In general, when D1 is excited (at a shorter, higher energy wavelength) it re-emits a photon at a longer wavelength (within D1's emission spectrum), and this re-emitted photon is in turn absorbed by D2 to produce an excited state. As D2 returns to its ground state, it emits a photon of light at a wavelength characteristic of its spectrum. In radiationless energy transfer, the reporter dipole interacts or resonates with the donor dipole where the energy level difference between the fluorophores corresponds to the quantum of excitation energy. In this process, the quantum, or exciton, is transferred, which raises the electron in the reporter to a higher energy state as the photo-excited electron in the donor returns to ground state. D1 transmits its excitation energy directly to D2 through the chain of linking atoms, without D1 ever releasing a photon. After D2 absorbs this energy it returns to its ground state while emitting a photon. Conditions include that the fluorescent emission spectrum of the energy donor overlap the absorption spectrum of the energy reporter, and that the donor and reporter transition dipole orientations must be approximately parallel. Energy transfer may be detected from an increase in reporter emission or a decrease in donor emission. In either case (direct or indirect) the result of such energy transfer is that D2 can be induced to emit a photon where the initial excitation light put into the system is of a wavelength that would not normally excite D2.
Notwithstanding the available fluorescent dyes and their application in various analytical methods including simple fluorescence, FET and FRET, it has remained desired to provide new and improved fluorescent dyes, FET and FRET dye couples, multiple-color energy transfer sets and methods of their use, particularly where no purification to remove excess fluorescent marker is required to achieve a satisfactory detection signal.